Download NLP-12 Engages Different UNC-13 Proteins to Potentiate Tonic and

1038 • The Journal of Neuroscience, January 21, 2015 • 35(3):1038 –1042
Brief Communications
NLP-12 Engages Different UNC-13 Proteins to Potentiate
Tonic and Evoked Release
X Zhitao Hu,1,2 Amy B. Vashlishan-Murray,1,2,3 and Joshua M. Kaplan1,2
1Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, 2Department of Neurobiology, Harvard Medical School,
Boston, Massachusetts 02115, and 3Department of Communication Sciences and Disorders, Emerson College, Boston, Massachusetts 02116
A neuropeptide (NLP-12) and its receptor (CKR-2) potentiate tonic and evoked ACh release at Caenorhabditis elegans neuromuscular
junctions. Increased evoked release is mediated by a presynaptic pathway (egl-30 G␣q and egl-8 PLC␤) that produces DAG, and by DAG
binding to short and long UNC-13 proteins. Potentiation of tonic ACh release persists in mutants deficient for egl-30 G␣q and egl-8 PLC␤
and requires DAG binding to UNC-13L (but not UNC-13S). Thus, NLP-12 adjusts tonic and evoked release by distinct mechanisms.
Key words: C. elegans; CCK; Munc13; neuropeptide; NLP-12; UNC-13
Introduction
To become fusion competent, synaptic vesicles (SVs) must physically attach to the plasma membrane (termed docking) and must
undergo a process termed priming (Verhage and Sorensen,
2008). Docking and priming are both mediated by the SNARE
proteins. Primed vesicles are thought to consist of docked SVs
containing partially assembled trans-SNARE complexes (Xu et
al., 1999). Several SNARE binding proteins regulate SV docking
and priming. UNC-10/RIM, UNC-13/Munc13, and UNC-31/
CAPS promote docking and priming while Tomosyn inhibits
both processes (Gracheva et al., 2006, 2007, 2008; McEwen et al.,
2006; Weimer et al., 2006; Hammarlund et al., 2007). Collectively, these studies suggest that Munc13 (and other priming factors) stimulate exocytosis by promoting SV docking and the
initial assembly of trans-SNARE complexes.
SV priming factors are extensively regulated by second messengers (Verhage and Sorensen, 2008). For example, all Munc13
proteins have binding sites for DAG (the C1 domain), calcium
(C2B), and calmodulin (Betz et al., 1998, 2001; Shin et al., 2010;
Lipstein et al., 2012). Mutations that block DAG binding to
Munc13’s C1 domain block potentiation of synaptic transmission by synthetic DAG ligands (phorbol esters; Lackner et al.,
1999; Rhee et al., 2002; Basu et al., 2007). Similarly, mutations
that block calcium and calmodulin binding to Munc13 alter
short-term plasticity (Shin et al., 2010; Lipstein et al., 2013).
Thus, treatments altering individual second messengers adjust
Received July 11, 2014; revised Nov. 21, 2014; accepted Nov. 26, 2014.
Author contributions: Z.H., A.V.-M., and J.M.K. designed research; Z.H. and A.V.-M. performed research; Z.H.,
A.V.-M., and J.M.K. analyzed data; Z.H., A.V.-M., and J.M.K. wrote the paper.
This work was supported by a National Institutes of Health research grant (GM54728 to J.K.). We thank members
of the Kaplan lab for critical comments on this manuscript. Some strains were provided by the C. elegans Genetics
Stock Center.
The authors declare no competing financial interests.
Correspondence should be addressed to Joshua M Kaplan, Department of Molecular Biology, Massachusetts
General Hospital–Simches Research Building, 7th Floor, 185 Cambridge Street, Boston MA 02114. E-mail:
[email protected].
DOI:10.1523/JNEUROSCI.2825-14.2015
Copyright © 2015 the authors 0270-6474/15/351038-05$15.00/0
Munc13 priming activity and neurotransmitter release. In contrast, endogenous neuromodulators simultaneously activate
multiple second messengers. For example, GPCRs activate protein kinases, Rac and Rho GTPases, as well as phospholipases.
Thus, natural neuromodulators may have more complex effects than treatments designed to manipulate individual second messengers.
The Caenorhabditis elegans neuromuscular junction (NMJ)
has been used as a model to address these questions. Transmission at this synapse is mediated by graded release of ACh,
whereby release varies with the strength of depolarization (Liu et
al., 2009). When activity is low, transmission consists of mEPSCs
that are evoked by fusion of a single SV (Liu et al., 2005), hereafter
designated tonic release. Forced depolarization of motor neurons evokes the synchronous release of several hundred SVs.
Prior genetic studies suggest that egl-30 G␣q and its target
(egl-8 PLC␤) enhances transmission at NMJs (Hajdu-Cronin
et al., 1999; Lackner et al., 1999; Miller et al., 1999). These
studies used behavioral (not electrophysiological) assays; consequently, it remains unclear how egl-30 G␣q and egl-8 PLC␤
alter ACh release.
We previously showed that a neuropeptide (NLP-12) and its
receptor (CKR-2) potentiate tonic and evoked ACh release at
NMJs (Hu et al., 2011). Here we show that potentiation of evoked
release requires activation of egl-30 G␣q and egl-8 PLC␤, and that
distinct UNC-13 proteins mediate the resulting potentiation of
tonic and evoked ACh release.
Materials and Methods
Strains. Animals were cultivated at 20°C on agar nematode growth media
seeded with OP50 bacteria. The following strains were used in this study:
wild-type N2 bristol, DA1084 egl-30(ad806), JT47 egl-8(sa47), KP6901
unc-13(s69), KP7451 nuEx1677 [Pacr-2::EGL-30];egl-30(ad806),
KP7447 nuEx1673 [Pacr-2::EGL-8];egl-8(sa47), KP6893 nuEx1515
[Psnb-1::UNC-13L];unc-13(s69), KP6899 nuIs46 [Punc-13::UNC13S::GFP];unc-13(s69), KP6893 nuEx1515 [Psnb-1::UNC-13L(H699K)];
unc-13(s69), and KP6899 nuIs52 [Punc-13::UNC-13S(H348K)::GFP];
unc-13(s69).
Hu et al. • Differential Control of Tonic and Evoked Release
EGL-30 G␣q is required for synaptic potentiation by NLP-12
NLP-12 and EGL-30 G␣q are both required for synaptic potentiation by aldicarb, consistent with the idea that NLP-12 potentiates release via activation of EGL-30. We did two further
experiments to test this idea. First, we analyzed tonic release in
nlp-12 eat-16 and eat-16; ckr-2 double mutants. The enhanced
mEPSC rate exhibited by eat-16 single mutants was significantly
reduced in both nlp-12 eat-16 and eat-16; ckr-2 double mutants
Control
+ Aldicarb
50 pA
0.5 nA
200 ms
10 ms
+ Aldicarb
Control
E
B
**
2
1
9
8
9
0
_ +
Rescue: WT
egl-30
C
10
8
+
egl-8
***
***
50
0
Aldicarb:
_
10 24
9
_
_
+
WT
9
5
9
_
+
+
egl-30 egl-8
F
***
50
***
mEPSC rate (Hz)
*
**
**
0 6 6 6
_ +
Rescue: WT
egl-30
9
_
6
+
egl-8
Tonic potentiation
(fold change)
Aldicarb potentiation of release is decreased in egl-30 G␣q
and egl-8 PLC␤ mutants
The effects of NLP-12 on release are assessed by recording EPSCs
following treatment with a cholinesterase inhibitor, aldicarb (Hu
et al., 2011). Aldicarb induces body muscle contraction, which
enhances NLP-12 secretion from a stretch sensing neuron (DVA;
Hu et al., 2011). Following aldicarb treatment, the mEPSC rate
and the total synaptic charge of evoked responses were both approximately doubled (Fig. 1 A, B,D–F ). The effects of aldicarb on
tonic and evoked release are eliminated in mutants lacking
NLP-12 and in those lacking an NLP-12 receptor (CKR-2; Hu et
al., 2011).
CKR-2 receptors are coupled to G-proteins containing a G␣qsubunit (Janssen et al., 2008). Consequently, we tested the idea
that egl-30 G␣q and egl-8 PLC␤ are required for aldicarb-induced
potentiation. The aldicarb-induced increase in evoked synaptic
charge was eliminated in both egl-30 G␣q and egl-8 PLC␤ mutants, and this defect was rescued by constructs expressing the
corresponding genes in cholinergic motor neurons (using the
acr-2 promoter; Fig. 1 A, B). Similarly, restoring egl-30 G␣q and
egl-8 PLC␤ expression in motor neurons rescued mutant defects
in aldicarb-induced paralysis (Fig. 1C). Thus, egl-30 G␣q and
egl-8 PLC␤ are required for aldicarb-induced potentiation of
evoked ACh release, as would be predicted if the NLP-12 receptor
(CKR-2) was coupled to Gq (Janssen et al., 2008). Interestingly,
the aldicarb-induced increase in mEPSC rate was only modestly
reduced in egl-30 G␣q mutants and was unaffected in egl-8 PLC␤
mutants (Fig. 1D–F ), implying that distinct mechanisms mediate
NLP-12 potentiation of tonic and evoked release.
To further investigate EGL-30’s role in tonic release, we analyzed eat-16 RGS mutants (Fig. 2). EAT-16 has GTPase-activating
activity for EGL-30 G␣q (Hajdu-Cronin et al., 1999); consequently, eat-16 mutants can be used to assess the effects of enhanced EGL-30 activity. In eat-16 mutants, mEPSC rates were
significantly increased (Fig. 2 A, B). This effect was abolished in
egl-30 eat-16 double mutants (Fig. 2 A, B). Thus, increased
EGL-30 G␣q activity produces a corresponding increase in tonic
release.
D
Evoked potentiation
(fold change)
Results
A
% paralyzed
(@ 60mins)
Constructs and transgenes. Transgenic strains were isolated by microinjection of various plasmids using either Pmyo-2::NLS-GFP (KP#1106)
or Pmyo-2::NLS-mCherry (KP#1480) as a coinjection marker. Integrated
transgenes were obtained by UV irradiation and were out-crossed at least
six times. EGL-30 (M01D7.7a; wormbase.org) or EGL-8 (B0348.4a;
wormbase.org) cDNAs were expressed in cholinergic motor neurons
using the acr-2 promoter.
Locomotion assays. For aldicarb paralysis, between 18 and 25 young
adult worms were transferred to plates containing 1.5 mM aldicarb and
assayed for paralysis as described previously (Nurrish et al., 1999).
Electrophysiology. Electrophysiology was done on dissected adults as
previously described (Richmond and Jorgensen, 1999; Hu et al., 2011,
2013). For aldicarb exposure, a single adult was transferred to a plate
containing 1 mM aldicarb for 60 min before the dissection. Statistical
significance was determined using a two-tailed Student’s t test.
J. Neurosci., January 21, 2015 • 35(3):1038 –1042 • 1039
2
ns
*
**
1
7
9
0
_ +
Rescue: WT
egl-30
24
9
_
5
+
egl-8
Figure 1. EGL-30/G␣q and EGL-8/PLC␤ are required for aldicarb-induced potentiation of
evoked release. Mutations inactivating EGL-30/G␣q and EGL-8/PLC␤ prevent aldicarb potentiation of evoked ACh release but have little effect on potentiation of tonic release. Evoked EPSCs
(A, B) and mEPSCs (D–F ) were recorded from adult body wall muscle, with (blue) and without
(black) a 60 min aldicarb treatment. Averaged evoked EPSCs (A) and representative mEPSC
traces (D) are shown. Summary data for evoked (B) and tonic (E, F ) release are shown for
wild-type, egl-30, and egl-8 mutants. Rescue indicates mutant animals containing a transgene
expressing the indicated gene in cholinergic motor neurons. C, Aldicarb-induced paralysis is
compared for the indicated genotypes. Statistically significant differences (***p ⬍ 0.001,
**p ⬍ 0.01, *p ⬍ 0.05, and ns not significant), the number of animals analyzed (B, E, F ), and
the number of replicate experiments (C) are indicated. Error bars indicate SEM.
(Fig. 2 A, B). The mEPSC rate of nlp-12 and ckr-2 single mutants is
indistinguishable from wild-type controls (Hu et al., 2011). Thus,
inactivating NLP-12 and CKR-2 decreased mEPSC rate only
when EGL-30 G␣q activity was enhanced (in eat-16 mutants).
Second, we analyzed aldicarb-induced paralysis of double mutants. As previously reported, both ckr-2 and egl-30 single mutants are resistant to aldicarb-induced paralysis; however,
additive effects on aldicarb sensitivity were not observed in egl30; ckr-2 double mutants (Fig. 2C). Together, these results support the idea the NLP-12, CKR-2, and EGL-30 G␣q act together
to potentiate ACh release.
Aldicarb-potentiated tonic release is mediated by UNC-13L
Activation of EGL-8 PLC␤ stimulates PIP2 hydrolysis, producing
the second messengers DAG and IP3. Prior studies suggested that
DAG binding to UNC-13/Munc13 promotes ACh release in C.
elegans and glutamate release in rodent neurons (Lackner et al.,
1999; Rhee et al., 2002). For this reason, we tested the idea that
UNC-13 is required for aldicarb-induced potentiation of ACh
release.
The unc-13 gene encodes two isoforms (UNC-13S and L),
which have different N-terminal domains but share a 1200 aa
Hu et al. • Differential Control of Tonic and Evoked Release
1040 • J. Neurosci., January 21, 2015 • 35(3):1038 –1042
A
A
UNC-13S
UNC-13S + A
UNC-13L
UNC-13L + A
UNC-13L(H699K)
UNC-13L(H699K) + A
WT
+ Aldicarb
Control
% paralyzed
(@ 60 mins)
mEPSC rate (Hz)
*
50
10
6
7
7
7
egl
-30
W
eat T
-16
eat
nlp
-16
-12
eat
-16
eat
-16
;ck
r-2
0
0
50
ns
0
9
9
9
9
Figure 2. Increased EGL-30 G␣q activity enhanced tonic release. A, B, The eat-16 RGS mutants have enhanced EGL-30 activity (Hajdu-Cronin et al., 1999) and a corresponding increase in
mEPSC rate. This effect was abolished in egl-30 mutants, and was diminished in nlp-12 and
ckr-2 mutants. Representative traces (A) and summary data (B) for mEPSCs are shown. C, The
egl-30 and ckr-2 single mutants were both resistant to aldicarb-induced paralysis but double
mutants did not exhibit additive defects. These results suggest that NLP-12 stimulates ACh
release by activating EGL-30. Statistically significant differences (***p ⬍ 0.001, **p ⬍ 0.01,
*p ⬍ 0.05, and ns, not significant), the number of animals analyzed (B), and the number of
replicate experiments (C) are indicated. Error bars indicate SEM.
C-terminal domain (Kohn et al., 2000; Hu et al., 2013). To determine which UNC-13 protein is required for NLP-12’s effects on
tonic release, we analyzed aldicarb potentiation of mEPSC rate in
unc-13(s69) mutants that express either UNC-13L or S transgenes (Fig. 3). Aldicarb-treatment increased the mEPSC rate of
UNC-13L-rescued animals and wild-type controls to similar levels, and this effect was eliminated by a mutation (H699K) that
disrupts DAG binding to UNC-13L (Fig. 3A–C; Betz et al., 1998).
In contrast, aldicarb had no effect on the mEPSC rate of UNC13S-rescued animals (Fig. 3A–C). Thus, NLP-12’s effects on tonic
release require DAG binding to UNC-13L.
Aldicarb potentiates evoked release mediated by both UNC13L and S
Which UNC-13 protein potentiates evoked release? Transgenes
expressing either UNC-13S or UNC-13L partially rescue the
baseline-evoked EPSC defect of unc-13(s69) mutants (Fig. 4 A, B),
consistent with our prior study (Hu et al., 2013). Partial rescue is
expected because the wild type-evoked response is a composite of
both UNC-13S and UNC-13L-mediated release (Hu et al., 2013).
Aldicarb potentiation of evoked release in UNC-13S- and UNC13L-rescued animals was similar to that in wild-type controls
(Fig. 4A–C). Potentiation of evoked responses was eliminated by
C1 domain mutations that disrupt DAG binding to UNC-13S
10 24
15 13
WT
S
6
8
7
C
7
***
2
1
0
L L(H/K)
UNC-13
***
24
13
WT S
8
7
L L(H/K)
UNC-13
Figure 3. Aldicarb potentiation of tonic release required DAG binding to UNC-13L but not
UNC-13S. Aldicarb potentiated tonic release in animals expressing UNC-13L but not in those
expressing UNC-13S. Potentiation of tonic release was blocked by a mutation that prevents DAG
binding to UNC-13L (H699K). UNC-13S or UNC-13L transgenes were expressed in unc-13(s69)
mutants. mEPSCs were recorded from adult body wall muscle of the indicated genotypes, with
(blue) and without (black) a 60 min aldicarb treatment. Representative traces (A), average
mEPSC rates (B), and aldicarb potentiation of mEPSC rates (C) are shown. Statistically significant
differences (***p ⬍ 0.001 and ns, not significant) and the number of animals analyzed are
indicated. Error bars indicate SEM.
A
UNC-13S
UNC-13S(H348K)
UNC-13L
UNC-13L(H699K)
0.5 nA
10 ms
+ Aldicarb
Control
B
Charge transfer (pC)
*** **
100
ns
50
200 ms
C
WT
egl
-30
c
kr
egl
-30 -2
;ck
r-2
B
***
C
***
40
***
0
ns
9
9
WT
10 24
10 8
6 9
6 6
+
H/K
+
H/K
UNC-13S
D
*
ns
20
**
Evoked potentiation
(fold change)
50 pA
***
Charge transfer
nlp-12 eat-16
mEPSC rate (Hz)
B
egl-30 eat-16
50 pA
200 ms
Tonic potentiation
(fold change)
eat-16
UNC-13L
2
***
1
0
9 24
8
9
6
WT + H/K + H/K
S
L
1.0
0.5
0
UNC-13S
10 20 30 40
Time (ms)
UNC-13L
10 20 30 40
Time (ms)
UNC-13L
H699K
10 20 30 40
Time (ms)
Figure 4. DAG binding to UNC-13S and L potentiated evoked release. Aldicarb-potentiated
evoked release was analyzed in animals expressing UNC-13S or UNC-13L. For both UNC-13
proteins, potentiation of evoked release was blocked by mutations that prevent DAG binding.
UNC-13 transgenes were expressed in unc-13(s69) mutants. Averaged evoked responses (A)
and summary data (B, C) are shown. D, The time course of charge transfer during evoked
responses is compared. The number of animals analyzed is indicated for each genotype. Statistically significant differences (***p ⬍ 0.001, **p ⬍ 0.01, *p ⬍ 0.05, and ns, not significant)
and the number of animals analyzed are indicated. Error bars indicate SEM.
(H348K) or UNC-13L (H699K; Betz et al., 1998; Nurrish et al.,
1999; Fig. 4A–C). Thus, NLP-12 potentiates evoked release via
DAG binding to both UNC-13 proteins.
We previously showed that the kinetics of evoked release mediated by UNC-13L is faster than that mediated by UNC-13S (Hu
et al., 2013). To determine whether aldicarb alters release kinet-
Hu et al. • Differential Control of Tonic and Evoked Release
ics, we analyzed the charge transfer kinetics of evoked responses.
Aldicarb treatment significantly slowed the charge transfer of
UNC-13L-mediated evoked responses and this effect was eliminated by the UNC-13L(H699K) mutation (Fig. 4D). In contrast,
aldicarb had no effect on the kinetics of UNC-13S-mediated
evoked responses (Fig. 4D). These results suggest that DAG binding to UNC-13L slows ACh release during evoked responses.
Discussion
Here we show that NLP-12 stimulates ACh release via DAG binding to UNC-13S and UNC-13L. These results identify an endogenous neuromodulator that potentiates release through the
PLC␤/UNC-13 pathway. UNC-13/Munc13 proteins have been
implicated in phorbol ester effects on secretion and in several
forms of short-term plasticity. Our results suggest that GPCRs
coupled to G␣q, and in particular neuropeptide receptors, potentiate transmission through changes in DAG liganding of UNC13. Consistent with this idea, CCK has been shown to stimulate
glutamate release in hippocampal neurons via a G␣q-coupled
receptor (Breukel et al., 1997; Deng et al., 2010). Because the
NLP-12 receptor (CKR-2) is most similar to mammalian CCK
receptors (Janssen et al., 2008), our results suggest that potentiation of neurotransmitter release by NLP-12/CCK-like neuropeptides is ancient and likely acts via changes in UNC-13 activity.
Neuropeptides are broadly expressed in the brain in both vertebrate
and invertebrate animals. Consequently, neuropeptide regulation of
UNC-13/Munc13 proteins provides a potential mechanism for
modulating circuit function and behavioral states.
Prior studies showed that different Munc13 isoforms mediate
different forms of short-term plasticity in rodent neurons (Rosenmund et al., 2002). Following high-frequency stimulus trains, synapses relying on Munc13-1 are depressed whereas those using
Munc13-2 are potentiated. UNC-13 proteins containing the C2A
domain (UNC-13L, Munc13-1, and ubMunc13-2) mediate a fast
form of release whereas UNC-13 proteins lacking the C2A domain
(UNC-13S and bMunc13-2) mediate slow release (Chen et al., 2013;
Hu et al., 2013; Zhou et al., 2013). Thus, differential expression of
Munc13 isoforms endows synapses with different patterns of release,
and different forms of plasticity.
Here we extend these studies by showing that NLP-12 potentiates tonic and evoked ACh release by distinct mechanisms. In
particular, our results indicate that different UNC-13 isoforms
regulate tonic (UNC-13L) and evoked (both UNC-13L and S)
release. These results provide genetic evidence that neuromodulators engage different UNC-13 proteins to regulate different
forms of release.
Aldicarb caused UNC-13L-mediated evoked release to become significantly slower and more prolonged, whereas it had no
effect on UNC-13S release kinetics. These results suggest that
DAG binding to UNC-13L loosens the coupling of primed SVs to
the calcium channel driving release. This could be mediated by
altered binding of UNC-13L to UNC-10/RIM or by changes in
the kinetics of calcium binding to UNC-13L primed SVs. Further
experiments are required to distinguish between these possibilities. These results suggest that neuromodulators like NLP-12
provide a means to adjust release kinetics.
NLP-12 potentiation of tonic and evoked release also differed
in their sensitivity to egl-30 G␣q and egl-8 PLC␤ mutations. The
egl-30(ad106) mutation is a partial loss of function; consequently,
residual aldicarb potentiation of tonic release in this mutant
could be mediated by residual EGL-30 activity. It is not possible
to test the effect of egl-30-null mutations, as these mutants are not
viable (Brundage et al., 1996). Alternatively, the residual poten-
J. Neurosci., January 21, 2015 • 35(3):1038 –1042 • 1041
tiation of tonic release in egl-30 mutants could be mediated by
other G␣-subunits. Further experiments are required to distinguish between these possibilities. DAG binding to UNC-13L is
required for potentiation of tonic release, as this effect is blocked
by the UNC-13L (H699K) mutation. Nonetheless, EGL-8 PLC␤null mutations had no effect on aldicarb-induced potentiation of
mEPSC rate. These results imply that DAG produced by other phospholipases potentiates tonic release.
How are tonic and evoked release differentially regulated? Aldicarb potentiates tonic release mediated by UNC-13L but not by
UNC-13S. UNC-13L has a C2A domain that is absent in UNC13S (Kohn et al., 2000; Hu et al., 2013). The C2A domain binds
UNC-10/RIM (Lu et al., 2006), localizing UNC-13L to the center
of the active zone (adjacent to the dense projection; Weimer et al.,
2006). UNC-13 proteins lacking C2A exhibit a more diffuse presynaptic distribution (Chen et al., 2013; Hu et al., 2013; Zhou et
al., 2013). RIM proteins bind voltage-activated calcium channels
(CaVs) thereby concentrating CaV channels at active zones (Han
et al., 2011; Kaeser et al., 2011; Graf et al., 2012; Müller et al.,
2012). Thus, our results suggest that aldicarb selectively promotes tonic release of SVs that are adjacent to presynaptic CaV
channels.
Several prior studies support the idea that different forms of
release are mediated by distinct sets of synaptic proteins. Mouse
DOC2 is required for spontaneous but not evoked neurotransmitter release (Groffen et al., 2010; Pang et al., 2011). Inactivating
synaptotagmin I or complexin decreases synchronous-evoked release but enhances spontaneous neurotransmitter release (DiAntonio and Schwarz, 1994; Littleton et al., 1994; Pang et al., 2006;
Hobson et al., 2011; Martin et al., 2011). Collectively, these results
suggest that the different patterns of release and different forms of
synaptic plasticity are dictated by the expression and function of
distinct synaptic proteins.
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